J48 Parasit Dis (June & December 2009) 33(1&2):48–53 J Parasit Dis (June & December 2009) 33(1&2):48–53 ORIGINAL ARTICLE J P D

Anthelmintic effi cacy of ethanolic shoot extract of Alpinia nigra on tegumental enzymes of buski, a giant intestinal parasite

Bishnupada Roy • Ananta Swargiary

Received: 13 August 2009 / Accepted: 28 August 2009 ©2009 Indian Society for Parasitology

Abstract Ethanolic shoot extract of Alpinia nigra, a properties and hence aqueous crude juice is traditionally used anthelmintic medicinal among consumed by the natives of the Hill states of north-east , the Tripuri tribes of north-east India, was tested in vitro Tripura in particular as a popular cure against intestinal worm to determine its anthelmintic effi cacy in gastrointestinal infections. The crude shoot extract of this plant has been trematode Fasciolopsis buski, using alterations in the reported to cause destruction of surface tegument leading to activity of vital tegumental enzymes viz. acid phosphatase paralysis and death of intestinal parasite (Roy and Tandon (AcPase), alkaline phosphatase (AlkPase) and adenosine 1999); the plant extract is also responsible for distortion and triphosphatase (ATPase). Live adult F. buski treated in vitro disorganization of cytoplasmic organelles and vacuolization with different concentrations of the plant extract showed of tegument in parasitic worms (Roy et al. 2009). signifi cant decline in the visible stain histochemically and The presence of several vital enzymes viz. acid enzyme activities. Quantitatively, the total enzyme activity phosphatase (AcPase), alkaline phosphatase (AlkPase), of AcPase, AlkPase and ATPase was found to be reduced adenosine triphosphatase (ATPase), 5´-nucleotidase (5′- by 45, 41 and 43%, respectively compared to the control. Nu) has been reported in the body tegument, and the The reference drug, praziquantel also showed more or various organs/tissues of many fl at worms (Kwak and less similar effect like that of the plant extract. The results Kim 1996; Pal and Tandon 1998; Kar and Tandon 2004; suggest that the tegumental enzymes of the parasite may Lalchhandama et al. 2008). All these enzymes which are be an important target of action for active component(s) of involved in various metabolic processes, are also believed A. nigra, which appears to act transtegumentally. to be involved in absorption and/or digestion in the parasite (Roy 1982; Poljakoiva-Krustena et al. 1983). Many Keywords Alpinia nigra, Anthelmintic, Fasciolopsis buski, commercial like parbendazole, piperazine Tegumental enzymes adipate, phenothiazine, diethiazine, diethylcarbamazine, centperzine, tetramisole and levamisole alter the metabolism and disrupt mitochondrial energy formation results in Introduction decrease in ATP levels (Agarwal et al. 1990; Aggarwal et al. 1992; Vinaud et al. 2009). Activities of many of these Alpinia nigra (Gaertn) Burtt (Family Zingiberaceae) is a enzymes are reported to be altered by crude ethanolic extract medicinal plant, the shoot of which is considered to have of traditionally used medicinal too, like vestita, Acacia oxyphylla, Millettia pachycarpa (Tandon B. Roy • A. Swargiary and Das 2007; Lalchhandama et al. 2007; Lalchhandama et Department of Zoology, North-Eastern Hill University, Shillong - 793 022, India al. 2008). Aqueous extract of Butea monosperma, Embelia e-mail: [email protected] ribes and Roltleria tinctoria causes reduction in both J Parasit Dis (June & December 2009) 33(1&2):48–43 49

AcPase and AlkPase activity in Paramphistomum cervi selected for histochemical and biochemical studies because in vitro (Chopra et al. 1991). of the early effect of the dose when compared with other In view of the functional signifi cance of tegumental low concentrations. enzymes in digestion or absorption in trematodes, the present investigation was aimed to study the effect of Histochemical localization of enzymes A. nigra ethanolic extract on the tegument of F. buski and enzymes viz. AcPase, AlkPase and ATPase. The following enzymes were investigated histochemically using duly processed frozen sections cut at a thickness of 10–15 μm in a Leica CM 1850 cryostat. Materials and methods AcPase activity was detected in cold formol-calcium- Preparation of the plant crude extract fi xed specimen following the modifi ed Lead nitrate method (Pearse, 1968), using sodium β-phosphoglycerate as the The edible fresh shoots of A. nigra were collected from substrate. A brownish precipitate indicates the sites of different places (villages) of Tripura, India. After washing AcPase. A modifi ed coupling azo-dye method as described gently with water the shoots were oven dried, grounded by Pearse (1968) was used for the detection of AlkPase by motor-driven grinder into powder form, and refl uxed in activity at room temperature (17–20°C). The brown color 90% alcohol for 12 hours at 60°C. After refl ux the solution was observed with fast violet B. For the localization of was fi ltered through Whatman fi lter paper No. 1 and the ATPase activity, the calcium method of Pearse (1968) was collected solution evaporated to dryness at 50°C to recover followed; ATP was used as the substrate and the enzyme dry powder, which was stored at 4°C till further use. activity was determined through observation of blackish brown deposit. Test parasites Biochemical assays Mature F. buski were collected from the intestine of freshly slaughtered pigs from the local abattoir in 0.9% phosphate Activities of AcPase and AlkPase were measured by buffer saline (PBS, pH 7.2). After washing in PBS, the fl ukes estimating the p-nitrophenol formation following the method were incubated at 37°C ± 1°C with different concentrations of Plummer (1988) with some modifi cations. A 10% (w/v) of the plant extract viz. 5, 10 and 20 mg/ml in PBS. tissue homogenate was prepared in sodium acetate buffer Praziquantel, a broad-spectrum anthelmintic drug, was used (125 mM, pH 4.5) and sodium glycine buffer (pH 10.5) for as the reference drug at similar concentrations. Control AcPase and AlkPase, respectively and centrifuged at 5,000 fl ukes were incubated in PBS alone. Three replicates for rpm for 20 minutes at 0 ± 2°C. The supernatant was taken each set of incubation medium were used and time taken for for the enzyme study. Using p-nitrophenyl phosphate as attaining paralytic state and death was recorded as described substrate the enzyme activity was measured by increase or earlier (Roy et al. 2009). Immediately after attained paralytic decrease in the optical density at 412 nm in a double beam state, the parasites incubated in 20 mg/ml of PBS were UV-visible spectrophotometer (Beckman Model-26).

Table 1 Histochemical localization of AcPase, AlkPase and ATPase in various body parts of Fasciolopsis buski Treatment mg/ml AcPase AlkPase ATPase T St Sm In T St Sm In T St Sm In Control (0.9% PBS) ++ ++++ ++++ +++ ++++ ++++ +++ +++ ++++ +++ +++ +++ A. nigra (20) ++ ++ - + ++ ++ + ++ ++ ++ + + Praziquantel (20) + + + + - + + + + + + +

T - Tegument; St - Sub-tegument; Sm - Somatic musculature; In - Intestine ++++ very intense activity, +++ intense activity, ++ moderate activity, + mild activity, - no activity. 50 J Parasit Dis (June & December 2009) 33(1&2):48–53

Table 2 Biochemical effects of shoot-extract of A. nigra and praziquantel on F. buski Treatment Enzyme activities (Total and specifi c) % decrease mg/ml AcPase AlkPase ATPase AcPase AlkPase ATPase Control 17.30 ± 0.26 37.40 ± 0.54 86.28 ± 0.54 (0.9% PBS) *1.70 ± 0.04 *3.93 ± 0.09 *8.25 ± 0.06

A. nigra (20) 9.44 ± 0.29 22.09 ± 0.28 49.17 ± 0.53 45.43 40.93 43.01 *0.97 ± 0.04 *2.53 ± 0.07 *5.06 ± 0.09

Praziquantel 7.33 ± 0.26 22.04 ± 0.27 44.85 ± 0.27 57.63 41.07 48.01 (20) *0.83 ± 0.02 *2.55 ± 0.04 *5.19 ± 0.04

Values are given as mean (±SE) from four replicates (n = 4). Total activity = formation of 1 μmol of product/h/g of wet tissue. *Specifi c activity = Activity/mg protein.

The ATPase activity was assayed by estimating the AlkPase and ATPase were observed in the tegument, sub- free phosphate released, following the method of Kaplan tegument, somatic musculature as well as intestine (Figs.

(1957) with ATP-Na2 as a substrate with little modifi cation, 1a, b; 2a, b and 3a, b). For all the enzymes highest intensity following Zaidi et al. (1981). A 10% tissue homogenate were observed mainly in tegument and sub-tegument was prepared in 0.1 M Tris-HCl buffer (pH 7.5). In a fi nal regions. In other regions like somatic musculature and volume of 1 ml reaction mixture containing 0.1 M Tris-HCl intestine also, high staining was noticed, though not similar buffer, 3 mM ATP-Na2, 30 mM MgCl2, 100 mM NaCl and to that in tegumental and sub-tegumental regions. 20 mM KCl, 0.2 ml of tissue homogenate was added and On exposure to the crude shoot extract of A. nigra and the mixture incubated for 1 hour. After incubation 1 ml 10% praziquantel, reduced activities of AcPase, AlkPase and TCA was added. The mixture was centrifuged for 10 minutes ATPase were observed (Table 1). The AcPase activity of the at a speed of 3,000–4,000 rpm in room temperature. Using somatic musculature and intestine were diminished, while the supernatant inorganic phosphate was determined by the mild visible staining was observed in the tegument and method of Fiske and Subba Row (1925). Similarly, blank sub-tegument regions of the plant extract-treated fl uke (Fig. tissue was prepared following the same method except the 1c, d). Almost all the regions of the praziquantel-treated TCA was added before the addition of tissue homogenate. fl ukes showed totally diminished staining activity for One unit of enzyme activity is defi ned as the amount AcPase (Fig. 1e, f). Reduction in the activity of AlkPase of product formed per h per g tissue. Specifi c activity is was also observed in the tegument, sub-tegument, somatic expressed as units/mg protein. The protein content of all the musculature and intestine of the crude plant extract- and tissue was estimated following the method of Lowry et al. praziquantel-treated fl ukes (Fig. 2 c, d, e, f); compared to (1951) using bovine serum albumin as a standard protein. other regions, less staining activity was observed in somatic musculature and intestinal regions. The ATPase activity was also found to be reduced in all the regions of the Alpinia- and Results praziquantel-treated parasites as compared to the controls (Fig. 3 c, d, e, f). The distribution and intensity of AcPase, AlkPase and In biochemical analysis also, all the enzymes in the ATPase in F. buski maintained as control, and treated with treated parasites showed highly reduced activity. AcPase, the ethanolic crude extracts of A. nigra and praziquantel AlkPase and ATPase were found to be reduced by 45.43, are presented in Table 1 and Figures 1–3. Quantitative 40.93 and 43.01%, respectively in the parasite treated with estimation of enzyme activity is depicted in Table 2. shoot-extract of the plant. Decreased enzyme activities In the control fl uke intense staining activities of AcPase, were also recorded in the fl ukes treated with praziquantel J Parasit Dis (June & December 2009) 33(1&2):48–43 51

Fig. 1 Acid phosphatase activity in F. buski, photographs of fresh Fig. 2 Alkaline phosphatase activity in F. buski, photographs of frozen sections fresh frozen sections a. Control (25×) a. Control (25×) b. Enlarged view of control (100×) b. Enlarged view of control (100×) c. Shoot extract of A. nigra treated section (25×) c. Shoot extract of A. nigra treated section (25×) d. Enlarged view of A. nigra treated section (100×) d. Enlarged view of A. nigra treated section (100×) e. Praziquantel treated section (25×) e. Praziquantel treated section (25×) f. Enlarged view of praziquantel treated section (100×) f. Enlarged view of praziquantel treated sections (100×)

(7.33 ± 0.26/0.83 ± 0.02, 22.04 ± 0.27/2.55 ± 0.04 and intensity of AcPase, AlkPase and ATPase were observed in 44.85 ± 0.27/5.19 ± 0.04, respectively). The enzyme the tegument of F. buski. Similar to the present observation, activities in the untreated control fl ukes were observed to be in a number of trematode and cestode parasites these much higher than the treated parasite (Table 2). phosphatases have been detected both histochemically and biochemically, and found to be closely associated with the tegument, sub-tegument, somatic musculature and gut Discussion (Leon et al. 1989; Pappas 1991; Pal and Tandon 1998; Lalchhandama et al. 2007, 2008). Anthelmintic drugs are known to enter target parasites AcPase and AlkPase are found to be involved in the by oral ingestion or by tegumental diffusion or sometime uptake of certain nutrients, glycogen and lipoprotein in by combination of both routes (Thompson et al. 1993; various helminth parasites (Sharma 1976). Differences in the Thompson and Geary 1995). Among trematode parasites the activity of these phosphatases have been detected in a large function of nutrient uptake, osmoregulation and digestion number of helminth parasites in relation to their difference is performed by tegument also in addition to intestine in pH values (Park and Seo 1968). Following the treatment (Meaney et al. 2002, 2004). of , the active component of Flemingia vestita, Consequently, it has been suffi ciently accounted that one the tegumental enzymes like AcPase, AlkPase, ATPase of the hallmark effects of any anthelmintic is destruction of and 5΄-nucleotidase of Raillietina echinobothrida were trematode surface (Roy and Tandon 1996, 1999; McKinstry found to be decreased many fold (Pal and Tandon 1998). et al. 2003; Xiao et al. 2004; Rivera et al. 2004). Changes in the aforementioned enzymes have also been In the present investigation high abundance and observed in F. buski following the treatment with genistein 52 J Parasit Dis (June & December 2009) 33(1&2):48–53

(Kar and Tandon 2004). Inhibition of AcPase activity by the plant extract as observed in the present investigation may suggest that the absorption and intracellular digestion of drugs may involve lysosomes (Colam 1971). Ultrastructural observations carried out on the surface tegument of F. buski also confi rmed its secretory and absorptive functions, a decrease in the enzyme activity is probably due to its leakage into the medium as a result of the disruption of the absorptive surface (Roy et al. 2009). In the present investigation both AcPase and AlkPase were found to be equally active in control F. buski. However, in the plant extract-treated fl uke, the AlkPase activity was revealed to be higher in the somatic musculature and intestine compared to AcPase, total activity of the enzyme was reduced by 40.93% after exposure to plant extract. A similar amount of reduction was also noticed in praziquantel-treated fl uke. In metacestodes of Echinococcus multilocularis, inhibition of the AlkPase activity by 23% and complete inhibition of glucose uptake were reported following treatment in vivo with isatin, a known phosphatase inhibitor, and the depletion in the enzyme activity was attributed to the failure of glucose Fig. 3 Adenosine triphosphatase activity in F. buski, photographs uptake (Delabre-Defayolle et al. 1989). However, more of fresh frozen sections a. Control (25×) than 2-fold increase in the AlkPase activity, observed in the b. Enlarged view of control (100×) praziquantel-treated Schistosoma mansoni was attributed to c. Shoot extract of A. nigra treated sections (25×) the drug-induced teguemntal damage exposing the normally d. Enlarged view of A. nigra treated sections (100×) concealed enzymes of the tegumental surface of the worm e. Praziquantel treated sections (25×) f. Enlarged view of praziquantel treated sections (100×) (Fallon et al. 1994). In the present study, high ATPase activity was observed in the tegument, sub-tegument, muscle layer and intestine of Reference the parasite. The ATPase activity was reduced by 43.01% in the fl ukes treated with the crude extract of A. nigra. A similar Agarwal A, Tekwani BL, Shukla OP, Ghatak S (1990) Effect of anthelmintics and phenothiazines on adenosine 5'- type of observation was recorded in F. buski treated with triphosphatases of fi larial parasite Setaria cervi. Indian J Exp crude extract of Flemingia vestita and its active principle, Biol, 28:245–248 genistein (Kar and Tandon 2004). Aggarwal R, Sanyal SN, Khera S (1992) Effect of anthelmintics The alteration observed in the activity of the phosphatase on phosphatases in . Acta Vet Hungarica, 40:243–249 enzymes of the shoot extract of A. nigra suggest that the Chopra AK, Sharma MK, Upadhyay VP (1991) Effect of ayurvedic tegumental enzymes of the parasite may be a plausible anthelmintics on phosphatase activity of Paramphistomum target of action for the plant product, which appears to act cervi. Indian J Parasitol, 43:65–69 transtegumentally. Colam JB (1971) Studies on gut ultrastructure and digestive physiology in Cyathostoma lari (Nematoda: Strongylida). Acknowledgements This study was supported by a project Parasitology, 62:273–283 grant from University Grants Commission, New Delhi to BR. Delabre-Defayolle I, Sarciron ME, Audin P, Gabrion C, Duniez T, Infrastructural support from DSA (UGC-SAP) program to the Paris J, et al. (1989) Echinococcus multilocularis metacestodes: Department of Zoology and UPE-Biosciences program to the biochemical and ultrastructural investigations on the effect School of Life Sciences, North-Eastern Hill University is gratefully of isatin (2–3 indoline dione) in vivo. J Antimicrob Agents acknowledged. Chemother, 23:237–245 J Parasit Dis (June & December 2009) 33(1&2):48–43 53

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